ABSTRACT

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-14C]butyrate and [1,4-13C1]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C4 intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.

Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H2 under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. 1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).

In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B12-dependent methylmalonyl-CoA mutase.

Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).

From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO2 acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).

Growth of M. sedula, cell harvest, and storage.M. sedula TH2 (DSM 5348) was grown in a 10-liter glass fermenter autotrophically and microaerobically on a chemically defined medium with a gas phase of 78% H2, 19% CO2, and 3% O2 at 75°C and pH 2.0 (generation time, 15 h) (31, 32). Heterotrophically grown cells were cultivated on the same mineral medium that included 0.5 g yeast extract per liter. The culture was slowly gassed with air (∼5 ml/min per liter culture medium). Growth was measured by determining the optical density at 578 nm (OD578 nm; light path = 1 cm). Cells were harvested at an OD578 of 0.25, immediately frozen, and stored at −80°C. The dry mass of washed cells (323 mg wet mass) derived from an exponentially growing 1-liter culture at an OD578 of 0.10 was determined after 60 h of freeze-drying as 56 mg dry mass. From the optical density, the dry mass and carbon content (50% of cell dry mass assumed) in samples of known optical density were calculated.

Labeling of growing cells.Starting at an OD578 of 0.09, [1,4-13C1]succinate was continuously pumped (Perfusor F; B. Braun, Melsungen, Germany) to the 10-liter culture growing autotrophically at 75°C with a generation time of 15 h. In addition, 185 kBq [1,4-14C]succinate was added as a tracer to quantify succinate metabolism. The pumping rate was 15 to 30 μmol succinate h−1, ensuring that less than 10% of the total cell carbon was derived from proffered succinate. During 22 h, a total amount of 0.6 mmol [1,4-13C1]succinate was added. After 1.5 generations, the cells were harvested at an OD578 of 0.25. In a second experiment, 366 kBq 4-hydroxy[1-14C]butyrate was added to a 2-liter culture at an optical density of 0.11 (resulting concentration of approximately 0.1 μM 4-hydroxybutyrate), and the culture was harvested at an optical density of 0.15 when most label was incorporated into the cell mass. The generation time was approximately 28 h. Radioactivity was determined in washed cells obtained by filtration through a 0.2-μm-pore size nitrocellulose filter and in the cell-free medium.

Fractionation of cell material.In the 14C labeling experiment, cell fractionation and protein hydrolysis were performed as described previously, with slight modifications (19). In the 13C labeling experiment, cells were disrupted by sonication. Three ml of H2O was added to 3 g (wet mass) of cells, and the suspension was treated several times (20 kHz for 20 s, 200 W) with a Branson-Sonifier W 450 (Branson Ultrasonics Corporation, Danbury, CT). After cell disruption, 3 ml of 70% (vol/vol) HClO4 was added to precipitate the protein. A centrifugation step (15 min, 48,000 × g, 4°C) followed after 30 min of incubation on ice. To remove substances of low molecular mass, the resulting pellet was washed with 10 ml of cold 0.5 M HClO4 three times, followed by centrifugation (5 min, 48,000 × g, 4°C). Ten ml of 0.5 M HClO4 was added to the pellet, and the solution was incubated at 70°C (20 min) to extract nucleic acids. Another centrifugation step (15 min, 20,000 × g, 4°C) followed this incubation. Nucleic acids remained in the supernatant, while proteins remained in the pellet. To extract lipids, 10 ml of a mixture of ethanol and diethyl ether (3:1, vol/vol) was added, and the suspension was incubated at 40°C for 1 h. A centrifugation step (15 min, 20,000 × g, 4°C) separated lipids (supernatant) and protein (pellet). After a wash (10 ml diethyl ether) and a centrifugation (15 min, 20,000 × g, 4°C) step, the protein pellet was dried at 80°C for 12 h.

Isolation of amino acids.For protein hydrolysis, 4 ml of 6 M HCl was added to the protein pellet, and this suspension was incubated in sealed and evacuated glass ampoules at 112°C for 24 h. HCl was removed by three evaporation cycles with the addition of 20 ml of H2O after each cycle. The pH was set to a value of 5.3 by adding 2 M NaOH, and the resulting solution was filtered. 13C-labeled amino acids were separated using chromatographic methods as described in Eisenreich et al. (15). 14C-labeled alanine, aspartate, and glutamate were isolated and quantified as described before (19).

NMR spectroscopy.1H and 13C nuclear magnetic resonance (NMR) spectra were recorded at 500.13 and 125.76 MHz, respectively, with a DRX500 spectrometer (Bruker Biospin, Rheinstetten, Germany) as described previously (14, 39). Briefly, 13C NMR spectra of the 13C-labeled compound under study and of natural-abundance material were recorded under the same experimental conditions. Integrals were determined for each 13C NMR signal, and the signal integral for each carbon atom in the labeled compound was referenced to that of the natural-abundance material, thus affording relative 13C abundances for each position in the labeled molecular species. Relative abundances were normalized by assigning a value of 1.1% to the carbon atom with the lowest 13C enrichment. The validity of this approach was confirmed for selected amino acids by the analysis of 13C coupling satellites in 1H NMR spectra.

Retrobiosynthetic data evaluation.From the carbon position of amino acids, the labeling of the central carbon metabolites was deduced (4, 14, 16, 19). For instance, the labeling of alanine directly reflects the labeling of pyruvate, its immediate biosynthetic precursor. However, valine, leucine, and isoleucine, which are partly derived from pyruvate, also contain reliable information and can be used. Thus, the labeling pattern was obtained from more than one source and averaged.

Enzyme assays.Cell extracts were prepared anaerobically by passing cell suspensions (1 g wet cell mass per 1 to 2 ml of 20 mM Tris-HCl, pH 7.8, containing 50 μg of DNase I ml−1) through a cooled French press cell at 137 MPa, followed by 1 h of 100,000 × g centrifugation at 4°C. Assays using auxiliary enzymes were conducted at 40°C; most other enzyme assays were performed at 65°C, except radioactive assays (75°C).

Pyruvate synthase and 2-oxoglutarate synthase were measured by following the exchange of 14C from 14CO2 into the C-1 carboxyl groups of the added 2-oxoacids, which was quantified as acid-stabile radioactivity by liquid scintillation counting (8, 55). Phosphoenolpyruvate (PEP) carboxylase was measured similarly by following the PEP-dependent incorporation of 14C from 14CO2 into acid-stable products (55). Enolase, phosphoglycerate mutase, and phosphoglycerate kinase were measured as described by Jansen et al. (40), with only minor modifications. Fructose 1,6-bisphosphate aldolase/phosphatase was measured by the method of Say and Fuchs (58). Isocitrate lyase and malate synthase were measured by the methods of Dixon and Kornberg (13). Succinyl-CoA synthetase and pyruvate-orthophosphate dikinase were measured according to Ramos-Vera et al. (55). Pyruvate-water dikinase was measured by the method of Eyzaguirre et al. (18). Glyceraldehyde 3-phosphate dehydrogenase was measured by the method of Zeikus et al. (70), but a pH of 7.5 instead of 8.1 was chosen. Succinate dehydrogenase was measured by the method of Herter et al. (27). Fumarate hydratase and malate dehydrogenase were measured as described by Berg et al. (8). Triosephosphate isomerase was measured spectrophotometrically at 365 nm by following the glyceraldehyde 3-phosphate-dependent oxidation of NADH (ε365, 3,400 M−1 cm−1) using glycerol 3-phosphate dehydrogenase as the auxiliary enzyme. The assay mixture (0.5 ml) contained 100 mM Tris-HCl (pH 7.9), 0.4 mM NADH, 5 mM MgCl2, 10 U of glycerolphosphate dehydrogenase, and cell extract. The reaction was started by adding 0.7 mM glyceraldehyde 3-phosphate [stock solution in 1 M 2-(N-morpholino)ethanesulfonic acid-NaOH, pH 6]. Malic enzyme was measured spectrophotometrically by following the l-malate- and MnCl2-dependent reduction of NAD(P)+ at 365 nm. The assay mixture (0.5 ml) contained 100 mM Tricine-KOH, pH 8.0, 5 mM dithiothreitol (DTT), 5 mM MnCl2, 1 mM NAD+ or NADP+, 10 mM l-malate, and cell extract. The reaction was inhibited by adding 5 mM EDTA. Succinic semialdehyde dehydrogenase was measured in a discontinuous assay by following by gas chromatography-mass spectrometry (GC-MS) with the formation of succinate from succinic semialdehyde upon the addition of NAD+, NADP+, or both. The assay mixture (4.5 ml) contained a buffer mixture of N-2-acetamido-2-hydroxyethanesulfonic acid (ACES), Tris, and ethanolamine buffers (pH 8.0) (17), 5 mM NAD(P)+, 1 mM MgCl2, 5 mM succinic semialdehyde, and 0.225 ml cell extract. Samples (1 ml) were taken after 0, 2, 5, and 10 min of incubation at 75°C, to which 0.1 ml of 50% (vol/vol) sulfuric acid was added. After centrifugation, the supernatant was mixed with 2 ml of methanol and 0.4 ml of 50% sulfuric acid and incubated for 12 h at room temperature to (di)methylate succinate and other organic acids. At this point, 1 ml of water and 0.5 ml of chloroform were added, and after thorough mixing and centrifugation, the organic phase was retrieved and analyzed by GC-MS. Succinate was quantified by using the peak area integral.

Analytical methods.The amount of 14C in liquid samples up to 200 μl was determined by liquid scintillation counting in a scintillation counter (Tri Carb 2100TR; Packard, Meriden, CT) using 3 ml of scintillation cocktail. The counting efficiency (75 to 85%) was determined via the channel ratio method. Amino acids were quantified with Ninhydrin reagent as described previously (19). Protein was determined by the Bradford method (9). Lactate derived from alanine was determined enzymatically (19). Succinate was quantified by GC-MS using a 6890Network GC system (Agilent Technologies, Santa Clara, CA) with a 30-m by 250-μm by 0.25-μm column connected to a quadrupole mass spectrometer with a 5973Network mass selective detector. The column (Agilent J&W Scientific, Santa Clara, CA) was coated with 5% phenylpolysiloxane and 95% dimethylpolysiloxane (sample size, 1 μl; flow rate, 1 ml min−1; pressure, 2.3 bar; temperature gradient at 0 to 3 min, 60°C; at 4 to 13 min, 60 to 180°C; and at 14 to 20 min, 280°C). The analyzed compounds were identified by the comparison of the characteristic fragmentation patterns to the database of the National Institute of Standards and Technology (Gaithersburg, MD, and Boulder, CO).

RESULTS

Incorporation of 4-hydroxy[1-14C]butyrate by autotrophically growing cells.This experiment aimed at testing whether 4-hydroxybutyrate, a key intermediate of the hydroxypropionate/hydroxybutyrate cycle (but not an intermediate of the established prokaryotic central carbon metabolism), was incorporated by growing cells into cellular building blocks. From the specific radioactivity of characteristic building blocks, one could infer the quantitative carbon flux starting from the proffered labeled precursor. M. sedula was grown autotrophically up to an OD578 of 0.11, and then approximately 0.1 μM 4-hydroxy[1-14C]butyrate (2 MBq μmol−1) was added. The incorporation of label into the cells was continuously monitored. It took approximately half a generation for the cells to take up most radioactivity (90%), at which time point the culture was harvested at an OD578 of 0.15. The residual (10%) labeled substrate may have been converted spontaneously to butyro-1,4-lactone, which cannot be metabolized. In the cell mass, 80% of the proffered label was recovered. Obviously, there was almost no loss of 14CO2; consequently, the substrate was not oxidized. Some minor loss of 14C as volatile 14CO2 was to be expected owing to decarboxylation reactions in biosynthesis, as discussed below. Due to the extremely low concentration, 4-hydroxybutyrate contributed only 0.3% to the carbon of newly synthesized cells. This guaranteed that the autotrophic carbon flux was not manipulated by the added organic tracer compound.

The cell material was fractionated, and 42% of the incorporated label was found in the protein fraction, 33% in the lipid and pigment fraction, and 12% in the nucleic acids fraction. This indicated that label from 4-hydroxy[1-14C]butyrate was incorporated into all macromolecules, and the amount of incorporated 14C roughly corresponded to the macromolecule contribution to the cell mass. This showed that 4-hydroxybutyrate, an odd intermediate, was converted into a common central metabolite, from which all cell carbon and building blocks were derived. From the hydrolyzed protein fraction, alanine, aspartate, and glutamate were isolated, and their specific radioactivity was determined. The ratio of the specific radioactivity of alanine (130 Bq μmol−1)/aspartate (147 Bq μmol−1)/glutamate (265 Bq μmol−1) was 1.0:1.1:2.0, indicating that 4-hydroxy[1-14C]butyrate gave rise to single-labeled pyruvate, single-labeled oxaloacetate, and double-labeled 2-oxoglutarate.

Retrobiosynthetic analysis.According to the proposed carbon fixation cycle (Fig. 1), one molecule of 4-hydroxy[1-14C]butyrate was converted to one molecule of [1-14C]acetyl-CoA and one molecule of unlabeled acetyl-CoA. The observed labeling of the three amino acids indicated that pyruvate (corresponding to alanine) and oxaloacetate (corresponding to aspartate) were built up from one labeled acetyl-CoA molecule plus one or two unlabeled bicarbonate molecules, respectively. Double-labeled 2-oxoglutarate (corresponding to glutamate) was synthesized from a single-labeled acetyl-CoA molecule and a single-labeled oxaloacetate molecule, which is in line with 2-oxoglutarate formation via citrate. This implies that almost no 14C was lost when isocitrate was oxidatively decarboxylated to 2-oxoglutarate. Thus, our data are fully consistent with the operation of the proposed carbon fixation cycle (Fig. 2). They support a metabolic scheme in which acetyl-CoA plus two molecules of bicarbonate are converted via 3-hydroxypropionate to succinyl-CoA, from which oxaloacetate, pyruvate, and 2-oxoglutarate then are derived (see Discussion).

Incorporation of radioactive label from 4-hydroxy[1-14C]butyrate by autotrophically growing M. sedula cells into metabolites of the carbon fixation cycle and into key metabolites of the central carbon metabolism. Labeled carbon is marked by a box. Only one incomplete turn of the carbon fixation cycle is shown for clarity reasons. The repetitive circulation would result in some scrambling of label, but it would not change the principal ratio of the labeling of pyruvate, oxaloacetate, and 2-oxoglutarate. Note that the second unlabeled acetyl-CoA molecule derived from the tracer 4-hydroxy[1-14C]butyrate is not shown. For the amount of label incorporated, see the text.

Incorporation of [1,4-13C1]succinate by autotrophically growing cells.To further investigate the central carbon metabolism in autotrophically grown cells, we grew M. sedula autotrophically in pure mineral medium under the continuous feeding of small amounts of single-labeled [1,4-13C1]succinate. The feeding rate was chosen such that maximally 10% of cell carbon could have been derived from the added organic substrate. Energy was gained by the oxidation of hydrogen with small amounts of oxygen. From these cells, protein was isolated and hydrolyzed, 10 amino acids were purified, and the 13C distribution within the carbon skeleton of the amino acids was determined by quantitative 13C NMR spectroscopy.

The observed 13C enrichment pattern is presented in Table 1. Many amino acids, e.g., glutamate or proline, showed high labeling mainly in the C-1 carboxyl but not in the C-5 carboxyl, whereas in aspartate or threonine both C-1 and C-4 carboxyl groups were 13C labeled. For comparison, the completely different labeling data of Chloroflexus aurantiacus and Thermoproteus neutrophilus also are given. These data, from earlier experiments, were obtained under the same experimental conditions (60, 61, 64).

Absolute 13C abundance of amino acids from M. sedula autotrophically grown in the presence of small amounts of [1,4-13C1]succinatea

Retrobiosynthetic analysis.The labeling patterns of these and other central intermediates were deduced by retrobiosynthetic analysis (Fig. 3, Table 2). Alanine, aspartate, and glutamate are formed by (trans)amination from pyruvate, oxaloacetate, and 2-oxoglutarate, respectively. Therefore, the labeling patterns of these amino acids directly reflected the patterns of pyruvate, oxaloacetate, and 2-oxoglutarate, respectively. Moreover, the labeling pattern of 2-oxoglutarate also was reflected by proline and the pattern of oxaloacetate, e.g., by threonine. Since the patterns of 10 amino acids were determined, the 13C enrichments of central intermediates could be calculated from different amino acids. The standard deviations shown in Table 2 provide information about the accuracy of the method and the reliability of the retrobiosynthetic approach.

Incorporation of label from [1,4-13C1]succinate into the central metabolites pyruvate, oxaloacetate, and 2-oxoglutarate. The synthesis of 2-oxoglutarate shown here assumes the functioning of (si)-citrate synthase. Labeled carbon atoms are marked by boxes. Compare the labeling pattern to those in Tables 1 and 2.

Average 13C enrichments of central metabolic pools in M. sedula grown autotrophically in the presence of small amounts of [1,4-13C1]succinatea

For simplicity, we will focus mainly on the labeling pattern of pyruvate, oxaloacetate, and 2-oxoglutarate, but the labeling patterns of the other metabolites are explained as well. Pyruvate, oxaloacetate, and 2-oxoglutarate showed high 13C labeling at C-1; 2-oxoglutarate was not labeled at C-5. This excludes the involvement of 2-oxoglutarate synthase in CO2 fixation; the reductive carboxylation of [1,4-13C1]succinate should have resulted in the formation of [2,5-13C1]2-oxoglutarate. The data rather favor the synthesis of 2-oxoglutarate through enzymes of an incomplete citric acid cycle (Fig. 1). The high labeling at C-1 of pyruvate indicates that this carbon atom did not derive directly from CO2, as one would expect if pyruvate synthase was active in CO2 fixation under these conditions. The labeling of oxaloacetate C-1 and, to a somewhat lesser extent, C-4 suggests that it was derived from succinate oxidation to oxaloacetate via malate. Some succinate may have been further metabolized via succinyl-CoA and 4-hydroxybutyrate, which would result in some scrambling of label. For a metabolic scheme, see Discussion.

Activities of enzymes that interconvert C4 and C3 compounds.The labeling studies corroborated the functioning of the proposed autotrophic carbon fixation cycle and the biosynthesis of different building blocks from central metabolites. These studies, however, did not provide an unambiguous answer to the question of how pyruvate and phosphoenolpyruvate, as well as the other central metabolites, are formed from intermediates of the cycle. There are only two likely starting molecules, succinyl-CoA and acetyl-CoA, as all other intermediates of the cycle cannot serve as universal cell carbon precursor molecules (Fig. 1 depicts intermediates). To address this question, we searched in cell extracts from autotrophically grown cells for the corresponding enzyme activities that could convert succinyl-CoA into central carbon precursor molecules. We first checked the genome of M. sedula (2) for candidate genes of the central carbon metabolism (Table 3). Indeed, they were found for most detected enzymes (Table 3).

For technical reasons, the assay temperature in most measurements was ≤65°C, which is below the optimal growth and cultivation temperature (75°C). All activities were detected at activity levels that were sufficiently high to explain the growth rate of the cells. None of the enzymes of the central carbon metabolism showed a dramatic regulation in response to heterotrophic growth.

Two enzymes could form succinate from succinyl-CoA (Fig. 1 and Table 3). Succinyl-CoA synthetase activity catalyzed the reversible ADP- and phosphate-dependent conversion of succinyl-CoA to succinate and ATP. Succinate was formed at even higher rates from succinic semialdehyde by NAD(P)+-dependent succinic semialdehyde dehydrogenase. Succinic semialdehyde is an intermediate of the autotrophic cycle being formed by the NADPH-dependent reduction of succinyl-CoA by succinyl-CoA reductase. The oxidation of succinate to oxaloacetate involves two dehydrogenases, succinate and malate dehydrogenases. Succinate dehydrogenase activity was membrane bound and tested with dichlorophenolindophenol as an artificial electron acceptor. Malate dehydrogenase [NAD(P)+] activity was high when measured in the direction of malate formation; this reaction was neither inhibited by EDTA nor dependent on Mn2+. In the direction of malate oxidation, some activity was observed with NADP+ only at alkaline pH; near neutral pH, this activity was almost zero. In contrast, malic enzyme (NAD+) activity was observed at neutral pH, which was dependent on Mn2+ and was inhibited by EDTA. This enzyme catalyzes the oxidative decarboxylation of malate, yielding pyruvate. An enzyme catalyzing pyruvate phosphorylation to PEP, pyruvate:water dikinase, also was present. No PEP carboxylase activity could be detected, yet GTP-dependent PEP carboxykinase activity was present. Usually, this enzyme is responsible for oxaloacetate conversion to phosphoenolpyruvate rather than for the reverse reaction.

Activities of enzymes of gluconeogenesis from PEP.All enzyme activities required to transform two molecules of PEP to one molecule of fructose 6-phosphate were present (Table 3). Glyceraldehyde 3-phosphate dehydrogenase preferred NADPH as the electron donor. Interestingly, this archaeon, like most Archaea, contained a bifunctional fructose 1,6-bisphosphate aldolase/phosphate that directly transforms two molecules of triosephosphate into fructose 6-phosphate and phosphate (58).

DISCUSSION

In this study, we addressed how central carbon precursor metabolites are drained off the hydroxypropionate/hydroxybutyrate cycle during the autotrophic growth of M. sedula. Our data show that most cell carbon is derived from succinyl-CoA. The reductive carboxylation of acetyl-CoA to pyruvate catalyzed by pyruvate synthase does not operate under the experimental conditions. This conclusion differs from the original suggestion that acetyl-CoA is the main precursor molecule (6); nevertheless, it is the key intermediate that is needed to build up succinyl-CoA. In addition, the labeling data perfectly support the functioning of the hydroxypropionate/hydroxybutyrate cycle and can hardly be explained by any other proposed metabolic scheme.

4-Hydroxy[1-14C]butyrate incorporation.We first consider the incorporation of label from C-1 of 4-hydroxybutyrate (Fig. 2). It was converted to two molecules of acetyl-CoA, one of which was labeled at C-1. A minor fraction of acetyl-CoA needs to be retained as immediate building block (2-oxoglutarate formed via citrate, lipids, etc). The major part is carboxylated to malonyl-CoA and further converted to succinate, taking up on its way two molecules of bicarbonate.

Succinyl-CoA is a branch point, and two routes lead away from its pool. The major part leads back to acetyl-CoA to regenerate the starting molecule of the cycle (Fig. 1). The rest is channeled to malate and oxaloacetate, from which C3 compounds are derived. Figure 2 shows the labeling observed after one turn of the cycle, which explains that the ratio of the label content in alanine, aspartate, and glutamate was approximately 1:1:2. Obviously, double-labeled 2-oxoglutarate was synthesized from single-labeled acetyl-CoA and single-labeled oxaloacetate via an incomplete citric acid cycle. If the cycle were complete, a substantial amount of labeled 14CO2 would have been lost, which was not observed.

[1,4-13C1]succinate incorporation.The data of the [1,4-13C1]succinate feeding experiment clearly reflect a situation in which most incoming labeled succinate was immediately used for making biosynthetic precursors (Fig. 3). At first sight this may be unexpected, since one would expect some scrambling of label. Scrambling is expected if succinate were in rapid equilibrium with succinyl-CoA, an intermediate of the autotrophic carbon fixation cycle. Fortunately, succinate conversion to succinyl-CoA obviously was minor, and succinate was pulled away, resulting in a rather distinct labeling pattern in central metabolites. If succinate was oxidized to oxaloacetate, equal label in C-1 and C-4 would be expected, and the ensuing decarboxylation of C4 intermediates to pyruvate or PEP would result in C-1-labeled C3 intermediates (pyruvate, PEP, and triosephosphates). This excluded a reductive carboxylation of acetyl-CoA to pyruvate, which then would have carried no label in C-1. The lower label content in C-4 of oxaloacetate may be explained by the reversibility of malic enzyme (acting on malate) and/or PEP carboxykinase (acting on oxaloacetate). The action of these enzymes would cause a partial loss of the labeled C-4 carboxyl group because of the incorporation of unlabeled CO2. The labeling of 2-oxoglutarate mainly at C-1 is consistent with its synthesis from oxaloacetate and acetyl-CoA via (si)-citrate synthase; (re)-citrate synthase would yield C-5-labeled 2-oxoglutarate (23).

Comparison of labeling pattern after [1,4-13C1]succinate incorporation in Metallosphaera, Thermoproteus, and Chloroflexus.The labeling patterns obtained with M. sedula are quite different from the patterns of either T. neutrophilus or C. aurantiacus (64). T. neutrophilus, an anaerobic crenarchaeon, uses the dicarboxylate/4-hydroxybutyrate cycle (34, 55, 56). This cycle involves pyruvate synthase and PEP carboxylase as carboxylating enzymes, and alanine, aspartate, and glutamate showed no 13C labeling in C-1, in contrast to M. sedula. Previously, the T. neutrophilus data were interpreted in terms of the operation of a reductive citric acid cycle (5, 59-61, 64). However, recently they were found to conform perfectly to the operation of the dicarboxylate/4-hydroxybutyrate cycle (55). C. aurantiacus uses the rather complex 3-hydroxypropionate bi-cycle for CO2 fixation, scrambling the label through its intermediates (27-30, 63, 64, 69).

Enzyme activities.The enzyme activities found in cell extract were consistent with the interpretation of the labeling experiments. Figure 4 shows a simplified metabolic scheme and the metabolic fluxes of the autotrophic pathway linked to the central carbon metabolism. Succinate forms the connecting link. One turn of the autotrophic carbon fixation cycle is required to generate one molecule of acetyl-CoA from two molecules of bicarbonate. Most cell carbon is indirectly derived from acetyl-CoA. Some acetyl-CoA serves as immediate precursor for building blocks. The majority, however, is converted to succinyl-CoA by an additional half turn of the cycle, which requires another two bicarbonate molecules. Succinyl-CoA then is drained off to provide the carbon skeleton for most biosynthetic precursors.

Metabolic fluxes in autotrophically grown M. sedula cells. The open arrows symbolize metabolic fluxes leading away from central carbon metabolites to building blocks. The numbers in the arrows indicate the percentages of all biosynthetic fluxes that derive from the central carbon precursors, such as acetyl-CoA, pyruvate, oxaloacetate, etc. The numbers in the ovals refer to the percentage of the individual fluxes of the carbon fixation cycle that has to supply 100% of all biosynthetic central precursor metabolites. Since the cycle has to generate one extra molecule of acetyl-CoA and also regenerates the starting molecule acetyl-CoA, the flux leading to acetyl-CoA sums up to 200% acetyl-CoA formation. The values have to be compared to the total inorganic carbon fixation rate. M. sedula grew autotrophically with a generation time of 15 h, which corresponds to a specific growth rate (μ) of 0.766 h−1, requiring a specific carbon fixation rate of 64 nmol·min−1·mg−1 protein. This estimation is based on the approved equation correlating the specific substrate (S) consumption (dS) per time unit (dt) to the growth rate μ, dS/dt = (μ/Y)·X. Y represents the established growth yield for bacterial cells of 1 g of dry cell mass formed per 0.5 g of carbon fixed. Note that this figure is independent of the growth substrate and depends solely on the fact that ∼50% of bacterial cell dry mass is carbon. X refers to 1 g cell dry mass, and it is a truism that in bacteria ∼50% of cell dry mass is protein; hence, 1 g of cell dry mass corresponds to ∼0.5 g of protein. Assuming that two carbon atoms are fixed in one turn of the cycle, the minimal specific activities of the enzymes of the autotrophic carbon fixation cycle that are required in vivo are about 32 nmol·min−1·mg−1 protein (compare to data in Table 3).

Interestingly, succinate most likely is formed from succinyl-CoA in two different ways, directly (by succinyl-CoA synthetase) or via a bypass from succinic semialdehyde (by succinic semialdehyde dehydrogenase). The enzyme outfit supports succinate oxidation to oxaloacetate and the oxidative decarboxylation of malate to pyruvate by malic enzyme. There may be two ways to synthesize PEP from C4 compounds, either indirectly from malate via pyruvate formation (by malic enzyme and subsequent phosphorylation by pyruvate:water dikinase) or directly from oxaloacetate (by PEP carboxykinase). Gluconeogenesis from PEP is conventional, with the exception of a bifunctional fructose 1,6-bisphosphate aldolase/phosphatase (58). The glyoxylate cycle also could be responsible for acetyl-CoA assimilation. However, isocitrate lyase activity could not be detected, and its putative gene was not found in the genome. In contrast, malate synthase is present. Its participation in pentose metabolism in Sulfolobales has been proposed recently (50).

Role of pyruvate oxidoreductase.There is no definitive answer to the role of oxygen labile pyruvate (and 2-oxoglutarate) oxidoreductases in autotrophic carbon fixation in all Sulfolobales. On one side, our labeling studies excluded that under the experimental conditions M. sedula uses these enzymes for carboxylation. On the other side, various putative genes for 2-oxoacid:acceptor oxidoreductases are present and M. sedula showed very low activities of 2-oxoglutarate and pyruvate oxidoreductases (Table 3). They were upregulated in autotrophically grown cells (35), whereas in Acidianus brierley they were downregulated (38). Stygiolobus azoricus, a strict anaerobic member of the Sulfolobales, possesses pyruvate oxidoreductase (but not 2-oxoglutarate oxidoreductase activity) (8); it is worth testing whether under anaerobic autotrophic conditions Stygiolobus uses this oxygen sensitive enzyme for reductive carboxylation of acetyl-CoA, as do the anaerobic Thermoproteales and Desulfurococcales (34, 55).

In contrast to the possible effect of oxic/anoxic conditions, we believe that energy sources less effective than H2, like metal sulfides (pyrite etc.), would not affect the cellular carbon flux. The generation of reduced ferredoxin would even require an energy-driven reverse electron flow. However, heterotrophic growth may demand decarboxylating 2-oxoacid oxidoreductases; this is owing to the fact that most Archaea do not contain 2-oxoacid dehydrogenase complexes but instead the ferredoxin-dependent oxidoreductases (25). This situation likely applies to the heterotrophic growth of Sulfolobales; heterotrophically grown Sulfolobus solfataricus P1 indeed contains ferredoxin and a 2-oxoacid:ferredoxin oxidoreductase with broad specificity (51).

Further supporting information.Auernik and Kelly presented a comprehensive transcriptome analysis of autotrophically, mixotrophically, and heterotrophically grown M. sedula cells that provided highly valuable supporting information (3). Our accompanying study on the missing enzymes in the autotrophic carbon fixation cycles in Crenarchaeota closes most gaps in the annotation of genes and in the characterization of the corresponding enzymes in M. sedula and T. neutrophilus (57).

ACKNOWLEDGMENTS

This work was supported by the Deutsche Forschungsgemeinschaft and the Hans-Fischer Gesellschaft Munich.

Thanks are due to Wolfgang Hüttel, Freiburg, Germany, for help with GC-MS.